Antimicrobial Effectiveness of Cellulose Based Fabrics Treated

247

Jelena Peran 1, Sanja Ercegović Ražić 1, Ivan Kosalec2, Flora Ziberi1 1University of Zagreb, Faculty of Textile Technology, Prilaz baruna Filipovića 28a, 10000 Zagreb, Croatia 2University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, 10000 Zagreb, Croatia Antimicrobial E! ectiveness of Cellulose based Fabrics treated with Silver Nitrate Solution using Plasma Processes Protimikrobna učinkovitost celuloznih tkanin, obdelanih z raztopino srebrovega nitrata v plazmi

Original Scienti" c Article/ Izvirni znanstveni članek Received/ Prispelo 08-2017 • Accepted/Sprejeto 08-2017

Abstract In order to obtain antibacterial properties, the possibility of deposition of silver particles from silver nitrate

(AgNO 3) solutions by plasma deposition process using argon as a carrier gas (PDP-Ar) was explored. Hexam- ethyldisiloxane and acrylic acid were used as precursors and were deposited by plasma enhanced-chemical vapor deposition (PE-CVD). The processes were carried out on lyocell and modal fabrics and antimicrobial effi cacy was determined on E. coli and S. aureus using time kill assay method. The results of minimal inhibitory concentration (MIC) show that higher antimicrobial effi cacy on E. coli is exhibited by the solution of (AgNO 3) in ethylene-glycol (0.066 µg/ml) rather than in absolute ethanol (0.265 µg/ml). For S. aureus, minimal inhibitory concentrations of AgNO 3 solutions in both absolute ethanol and ethylene-glycol as solvents are obtained at the same value (0.132 µg/ml). Overall, the best antibacterial eff ect for both modal and lyocell samples has been achieved against E. coli using treatments with precursors (AAC and HMDSO) and Ag-

NO 3 in ethylene-glycol as solvent, with prolonged incubation time. Keywords: cellulose fabrics, plasma processes, AgNO3, quantitative microbiological method, antibacterial eﬃ cacy

Izvleček Za doseganje protibakterijskih lastnosti je bila raziskana možnost nanosa srebrovih delcev iz raztopin srebrovega nitrata (AgNO 3) v plazmi, kjer je bil kot nosilni plin uporabljen argon (PDP-Ar). Kot prekurzorja sta bila uporabljena heksametildisiloksan in akrilna kislina, ki sta bila nanesena iz parne faze v plazmi (PE-CVD). Postopki so bili izve- deni na tkaninah iz liocelnih in modalnih vlaken, protimikrobna učinkovitost pa je bila določena na E. coli in S. aureus z metodo določanja preživelosti. Rezultati minimalne inhibitorne koncentracije (MIC) kažejo, da ima raztopi- na srebrovega nitrata v etilenglikolu (0,066 μg/ml) višjo protimikrobno učinkovitost na E. coli kot v absolutnem etanolu 0,265 μg/ml). Za S. aureus so bile MIC AgNO 3 raztopin v obeh topilih, absolutnem etanolu in etilenglikolu, dosežene pri enaki vrednosti (0,132 μg/ml). Na splošno je bil najboljši protibakterijski učinek proti E. coli s podaljša- nim inkubacijskim časom dosežen za vzorce iz modalnih in liocelnih vlaken, obdelanih s prekurzorji (AAC in HMDSO) in AgNO 3 v etilenglikolu kot topilu. Ključne besede: celulozne tekstilije, AgNO 3, kvantitativna mikrobiološka metoda, protibakterijska učinkovitost

Corresponding author/ Korespondenčna avtorica: Tekstilec, 2017, 60 (4), 247-253 assist. mag. ing. techn. text. Jelena Peran DOI: 10.14502/Tekstilec2017.60.247-253 E-mail: [email protected] 248 Antimicrobial Eﬀ ectiveness of Cellulose based Fabrics treated with Silver Nitrate Solution using Plasma Processes

1 Introduction It can be used as a metal, salt and nanoparticle [7]. Single mechanism of antimicrobial action is not Plasma technology and its application in the eld fully understood, but some mechanisms have been of textile technology are known as ecologically proposed that suggest that silver ion reacts with friendly processes. Textile materials exposed to DNA and RNA molecules within the cell wall of plasma undergo through chemical and physical microorganisms and prevents the replication. In transformations in the surface layer. Using di er- addition, a reaction of silver with thiol groups ent gasses and reagents in the process of plasma (–SH) inside the cell is assumed which causes pro- nishing of textile materials, a variety of functional tein deactivation and reduction of enzyme activity. properties can be obtained. ose include ame is causes changes in metabolism, prevents prop- resistance, antistatic, hydrophobic, antibacterial agation and ultimately leads to the destruction of properties etc. e ultimate e ect depends on many microorganism [8‒10]. Also, it is proposed that sil- more factors such as plasma type, type of materials, ver acts by binding to key functional groups of en- plasma parameters, treatment conditions and the zymes. Bacterial plasma or cytoplasmic membrane process used [1, 2]. are an important target site for silver ion, which In this study, the application of low-pressure plas- causes the release of K + ions from bacteria. Silver ma for the enhancement of surface properties of ions inhibit cell division and damage the cell enve- lyocell and modal bers and the achievement of lope and content of bacteria [11]. Antimicrobial antibacterial properties by deposition of silver par- textiles are used in medical technology, health, hy- ticles was explored. Natural materials are an excel- giene products, home textiles, water treatment sys- lent media for the growth of microorganisms, es- tems, as well as clothes for everyday use and per- pecially in humid and warm conditions. While sonal protection [4, 12]. many synthetic bers have good resistance against Physical sputtering and Plasma Enhanced Chemi- microbial attack, natural bers are more easily af- cal Vapor Deposition (PE-CVD) under low-pres- fected. Proteins in keratin bers and carbohydrates sure plasmas are dominant techniques used in tex- in cellulose bers can serve as a source of food and tile nishing [1]. In this study, a relatively new energy for various bacteria, fungi and molds [3]. method was used for the deposition of antibacteri- Bacteria are single-celled organisms that reproduce al agent called Direct Plasma Deposition Process very quickly in appropriate conditions. ey are (PDP) (Figure 1). classi ed as gram-positive and gram-negative and certain types are pathogenic and might cause seri- ous health problems [4]. Antimicrobial treatments are carried out in order to control the growth and reproduction of microorganisms. Agents can kill bacteria or fungi (biocides) or control the growth and spread of microbes (biostatics). Both prevent the spread of infection, allergic and respiratory problems as well as degradation of textile materials in the form of discolouration, staining, odors and degradation of the bers. ere are various antimicrobial agents used which di er in their chemical composition and antimicrobial activity. Quater- Figure 1: Reagent bottle for direct plasma deposition nary ammonium compounds, polyhexamethylene process biguanide, triclosan and some heavy metals can be used for biocidal purposes whereas chitosan and e process consists of reagent bottle with antibac- plant-derived bioactive agents can be used as bio- terial agent, which is from one side connected to the statics [5]. Many heavy metals such as silver, copper, supply of argon gas and the other side to plasma zinc and cobalt have excellent antimicrobial proper- system. Argon is used as a carrier gas, which assists ties at very low concentrations but silver is the most the transfer of reagent to the plasma system and on commonly used in the eld of textile industry [6]. the textile substrate.

Tekstilec, 2017, 60 (4), 247-253 Antimicrobial Eﬀ ectiveness of Cellulose based Fabrics treated 249 with Silver Nitrate Solution using Plasma Processes

2 Materials and methods 2.4 Antimicrobial eﬃ cacy of silver nitrate solutions

2.1 Textile samples and chemical agents Antimicrobial e$ cacy of AgNO 3 solutions was deter- Plain wave lyocell (CLY) and modal (CMD) fabrics mined by known broth microdilution technique, (Lenzing, Austria) were used in this study. ey which speci es minimum inhibitory concentration were industrially prepared (desized and scoured) (MIC), i.e. the minimum concentration of antimicro- and weaved at the textile company Čateks, Croatia. bial agent that inhibits the growth of a microorgan-

By dissolving AgNO 3 (Sigma Aldrich) in absolute ism a& er a speci c time of incubation. e method is ethanol (Carlo Erba) and ethylene-glycol (Fluka based on the dilution of the antimicrobial agent in Analytical), 0.1 M solutions were prepared and used the culture medium in microtiter plates, followed by for processing the fabrics in order to achieve anti- addition of inoculum. If there is no growth of micro- bacterial properties. Hexamethyldisiloxane, HMD- organism colonies, the concentration is the well rep- SO (Sigma Aldrich, ≥98.5%) and acrylic acid, AAC resents MIC. For this purpose, a series of 12 concen- –3 (Acros Organic, 99.5%) were used as precursors. trations of AgNO 3 was prepared from 4146.75 + 10 Applied bacterial species were Escherichia coli to 2.07 + 10 –3 µg/ml. MICs were determined for E. ATCC 10536 (gram-negative bacteria) and Staphy- coli and S. aureus a& er incubation at 37 °C for 24 loccocus aureus ATCC 6538 (gram-positive bacte- hours using 2,3,5- triphenyltetrazolium chloride ria). e strains were grown in Tryptic soy agar (TTC) as a redox indicator for determination of bac- (105458 Tryptic soy agar, Merck Millipore, Germa- teria viability. In the presence of bacteria, TTC was ny). Müeller-Hinton Broth (BBLTM Müeller-Hin- reduced to red coloured substance triphenyl forma- ton Broth – BD, USA) was used as a nutrient broth zan (TPF) and the colour change was directly pro- medium for broth microdilution technique. portional to the viable active cells. e change in colour was measured spectrophotometrically at 540 nm 2.2 Plasma treatments (Labsystems iEMS MF, type 1404, lter F6). Treatments were done using low-pressure plasma system type NANO LF-40 kHz, by Diener. To en- 2.5 Antimicrobial eﬃ cacy of treated samples sure enhanced binding of silver particles on tex- Antibacterial e$ cacy of treated materials was deter- tile surface, activation process was carried out for mined by time kill assay method. It is a quantitative

5 minutes using O 2 gas (purity of 99.99%, Messer) microbiological method that determines the under optimized process parameters – pressure number of colony forming units of microorganisms 0.34 mbar, power 300 W, frequency 40 kHz and gas a& er a speci c time of incubation of samples. In this ow 40 cm 3/min. e pretreatments with HMDSO way, the rate at which a microorganism is killed as a and AAC were conducted in order to enhance ad- function of time can be established. Treated samples sorption of silver particles onto cellulose surface us- (1cm x 1cm) were inoculated with 50 µl of microor- ing PE-CVD process. e process was conducted ganism suspension in sterile Petri dishes. e anti- for 20 minutes at pressure of 0.18 mbar and power of bacterial e ectiveness against E. coli and S. aureus

150 W. AgNO 3 solutions were applied on activated was investigated at the moment of contact of the and pretreated samples using PDP-Ar process and sample with a suspension of microorganisms ( t0) argon plasma as a carrier gas for deposition. e and a& er incubation time of 6 hours ( t1) and 18 process was carried out for 20 minutes at pressure hours ( t2) at 37 °C. Using sterile tweezers, inoculat- 1.5 mbar, gas ow 40 cm 3/min and power 150 W. ed samples were then transferred in test tubes, which had been pre- lled with 1 ml of saline. A& er 2.3 FE-Scanning electron microscopy analysis mixing with vibromixer (Vortex vibromixer Genius Field emission scanning electron microscopy (FE- 3, Ika, Germany) for 30 seconds, 100 µl of the solu- SEM) was conducted on untreated and plasma tion was transferred to an Eppendorf tube of 2 ml treated samples using Tescan SEM microscope that had been previously lled with 900 ml of saline (Mira 3 LMU). Before testing, the samples were solution. In this way, the solution was diluted 10 steamed by vaporized mixture of gold and palladi- times. is solution was then serially diluted and um under argon plasma. Micrographs were ob- the aliquots of the dilutions series were spread-plat- tained using 4kx, 7kx and 10kx magni cations. ed on an agar medium to allow for enumerating the

Tekstilec, 2017, 60 (4), 0-0 250 Antimicrobial Eﬀ ectiveness of Cellulose based Fabrics treated with Silver Nitrate Solution using Plasma Processes colonies of the bacteria. Tryptic soy agar (105458 For every concentration and time point, colonies Tryptic soy agar, Merck Millipore, Germany) was used of viable bacteria were counted and CFU/ml (col- as a nutrient medium and quanti cation of the ony forming units per ml) was calculated accord- number of colony forming units of microorganisms ing to Equation 1: was done a& er incubation at 37 °C in the dark for 24 hours. e process is shown schematically in Figure 2. CFU no. of colonies = log (1) ml 10 volume of culture plate dilution factor

e nal results are expressed as a percentage of inhibition in de ned time intervals.

3 Results and discussion

3.1 Morfological characteristics of plasma treated samples Figure 3 shows the obtained SEM micrographs of untreated and plasma treated lyocell and modal samples. Figure 2: Schematic representation of time kill assay e SEM micrographs of untreated lyocell and mo- method dal samples show smooth, clear surface of bers.

a) b) c)

d) e) f)

Figure 3: SEM micrographs of (a) untreated CLY and (b) CMD samples; (c) CLY treated with AAC and AgNO 3 solution in ethylene-glycol; (d) CMD treated with AAC and AgNO 3 solution in ethanol; (e) CLY treated with HMDSO and AgNO 3 solution in ethylene-glycol; (f) CMD treated with HMDSO and AgNO 3 solution in ethanol

Tekstilec, 2017, 60 (4), 247-253 Antimicrobial Eﬀ ectiveness of Cellulose based Fabrics treated 251 with Silver Nitrate Solution using Plasma Processes

On the surface of bers treated with precursors For bacteria E. coli minimal inhibitory concentra-

(AAC, HMDSO) and AgNO 3 solutions, signi cant tion of AgNO 3 solution in absolute ethanol is presence of precursors and silver particles over the 0.265 µg/ml and of AgNO 3 solution in ethylene- entire ber surface is evident which was deposited glycol 0.066 µg/ml. According to the obtained re- a& er surface activation with O 2 plasma. It con rms sults, higher antimicrobial e$ cacy on E. coli is ex- the e ectiveness of treatments performed using hibited by the solution of silver nitrate in plasma processes. ethylene-glycol, rather than in absolute ethanol.

e MIC value of AgNO 3 solution in ethanol and 3.2 Minimal inhibitory concentrations in ethylene-glycol for S. aureus is 0.132 µg/ml. of silver nitrate solutions From MIC values, it is visible that a higher con-

Antimicrobial e ectiveness of silver nitrate on the centration of AgNO 3 solution in ethylene-glycol is bacterial species E. coli and S. aureus was determined. needed for inhibition of S. aureus than for E. coli . MIC values are presented graphically in Figure 4 and Based on the obtained data it can be concluded Figure 5. e value of the absorbance, which indi- that silver nitrate is an excellent antimicrobial cates the activity of microorganisms, in relation to a agent that exhibits excellent antimicrobial e ec- series of concentrations of tested solutions are shown. tiveness even at very low concentrations. Although both solutions demonstrate great results, a more e ective solution for both bacterial species proved

to be AgNO 3 solution in ethylene-glycol. 3.3 Antimicrobial eﬃ cacy of treated samples e testing results of antibacterial e ectiveness of the treated samples for bacterial species E. coli and S. aureus are presented in Figures 6‒9. From the results, a positive e ect of all treated samples for tested bacterial species is visible and it grows with the incubation time. e untreated textile samples as well as those treated only with HMD- SO and AAC show slight antibacterial e ect in time. It suggests that the mentioned samples are not a suf- Figure 4: Graphical representation of antimicrobi- cient nutrient for large-scale growth and propaga- al e! ectiveness of AgNO 3 solution in absolute ethanol tion of tested bacterial strains in de ned time. (ET) and ethylene-glycol (EG) on bacteria E. coli e best antibacterial e ect for lyocell samples against E. coli was achieved a& er the treatment with

AgNO 3 solution in ethylene-glycol using HMDSO as a precursor. A reduction of 75.2% of E. coli colonies a& er 18 hours of incubation was achieved. A 61.8% reduction of E. coli was obtained a& er 18 hours of incubation with the treatment of lyocell

with AgNO 3 solution in ethylene-glycol and pre- treatment with AAC. For modal samples, the highest inhibition was at- tained a& er 18 hours of incubation a& er the treat-

ment with AgNO 3 solution in absolute ethanol both using AAC (68.2%) and HMDSO (69.1%) as precursors. In previous research [13], it was found that the ap- Figure 5: Graphical representation of antimicrobi- plication of HMDSO as a precursor enhanced the al e! ectiveness of AgNO 3 solution in absolute ethanol adsorption of silver particles onto cellulose pro- (ET) and ethylene-glycol (EG) on bacteria S. aureus viding higher antibacterial e ect. According to the

Tekstilec, 2017, 60 (4), 247-253 252 Antimicrobial Eﬀ ectiveness of Cellulose based Fabrics treated with Silver Nitrate Solution using Plasma Processes obtained results for E. coli , both AAC and HMD- stronger defense system against silver ions. Because SO proved to be suitable for the application as pre- of its greater resistance, the application of higher cursors using plasma process, with HMDSO as a concentration of agent should be considered. slightly more e$ cient agent.

Figure 8: Inhibition of S. aureus of treated CLY samples Figure 6: Inhibition of E. coli of treated CLY samples

Figure 9: Inhibition of S. aureus of treated CMD samples

Figure 7: Inhibition of E. coli of treated CMD samples 4 Conclusion e results of antibacterial e ect against S. aureus (Figures 8 and 9) are not so uniform for the tested According to the obtained results, the following samples and inhibition is lower. e results for two conclusions can be made: treated samples that exhibit maximum inhibition • Plasma treatment certainly contributes to success- (CLY/AAC, 6 hours of incubation and CMD/AAC/ ful adsorption of chemical agents on man-made

AgNO 3/EG, 18 hours of incubation) have a high cellulosic bers. degree of deviation and are inconsistent so they • From SEM micrographs, a signi cant presence of should be taken into account with caution and precursors is evident on the entire ber surface, double checked. Besides them, the highest reduc- which con rms the e ectiveness of the applied tion of S. aureus for both lyocell and modal sam- PE-CVD process. ples was achieved a& er the treatment with AgNO 3 • SEM micrographs also show the presence of sil- solution in ethylene-glycol and HMDSO as a pre- ver particles on a treated ber surface, which su- cursor (18 hours of incubation). Feng et al. [10] ggests the applied PDP-Ar process was conduc- conducted a study of antibacterial e ect of silver ted successfully. ions on E. coli and S. aureus and observed higher • Based on the data for minimal inhibitory concen- resistance of S. aureus than E. coli , which suggests a tration of AgNO 3 solutions, it can be concluded

Tekstilec, 2017, 60 (4), 247-253 Antimicrobial Eﬀ ectiveness of Cellulose based Fabrics treated 253 with Silver Nitrate Solution using Plasma Processes

that silver is an excellent antimicrobial agent that 5. ZHAO, Yan, XU, Zhiguang, LIN, Tong. Barrier exhibits great antimicrobial e ectiveness even at textiles for protection against microbes. In Anti- very low concentrations. microbial textiles . Edited by S. Gang. Cambridge :

• According to MIC values, AgNO 3 solution in Woodhead Publishing, 2016, pp. 225‒245, doi: ethylene-glycol proved to be a slightly more ef- 10.1016/B978-0-08-100576-7.00012-2. fective solution for both tested bacterial species. 6. SHAHIDI, Sheila, WIENER, Jakub. Antibacte- • Highest inhibition for lyocell samples against E. rial agents in textile industry. In Antimicrobial

coli was achieved a& er the treatment with AgNO 3 agents . Edited by Bobbarla, V. Rijeka : InTech, solution in ethylene-glycol using HMDSO as a 2012, pp. 387‒406, doi: 10.5772/46246. precursor a& er 18 hours of incubation (75.2%). 7. CHERNOUSOVA, Svitlana, EPPLE, Matthias. • Highest inhibition for modal samples against E. Silver as antibacterial agent: ion, nanoparticle,

coli was achieved a& er the treatment with AgNO 3 and metal. Angewandte Chemie International solution in absolute ethanol using HMDSO as a Edition , 2013, 52 (6), 1636‒1653, doi: 10.1002/ precursor a& er 18 hours of incubation (69.1%). anie.201205923. • Results of antibacterial e ect against S. aureus 8. YURANOVA, Tataina, RINCON, A.G., BOZZI, obtained by time kill assay are not so uniform for A., PARRA, S., PULGARIN, C., ALBERS, P., the tested samples and the inhibition is lower. KIWI, J. Antibacterial textiles prepared by RF- e reason may be a higher resistance of such plasma and vacuum-UV mediated deposition bacteria to silver ions. of silver. Journal of Photochemistry and Photobi- • Overall, both AAC and HMDSO proved to be su- ology A: Chemistry , 2003, 161 (1), 27‒34, doi: itable for the application as precursors using PE- 10.1016/S1010-6030(03)00204-1. CVD process, with HMDSO as a slightly more ef- 9. RAI, Mahendra, YADAV, Alka, GADE, Aniket. cient agent. Silver nanoparticles as a new generation of anti- microbials. Biotechnology Advances , 2009, 27 (1), Acknowledgement: " is work has been fully support- 76‒83, doi: 10.1016/j.biotechadv.2008.09.002. ed by research related to scienti# c grant “Antibacteri- 10. FENG, Qing Ling, WU, J., CHEN, G. Q., CUI, F. al properties of textile materials achieved by ecologi- Z., KIM, T. N., KIM, J. O. A mechanistic study cal treatments” (coordinator Assist. Prof. M. Somogyi of the antibacterial e ect of silver ions on Es- Škoc). " is work has been partially supported by cherichia coli and Staphylococcus aureus . Journal Croatian Science Foundation (IP-2016-06-5278). of Biomedical Materials Research , 2000, 52 (4), 662‒668, doi: 10.1002/1097-4636(20001215)52:4 References <662::aid-jbm10>3.0.co;2-3. 11. JUNG, Woo Kyung, KOO, Hye Cheong, KIM, 1. ZILLE, Andrea, OLIVEIRA, Fernando Ribeiro, Ki Woo, SHIN, Sook, KIM, So Hyun, PARK, SOUTO, Antonio Pedro. Plasma treatment in tex- Yong Ho. Antibacterial activity and mechanism tile industry. Plasma Processes and Polymers , 2015, of action of the silver ion in Staphylococcus au- 12 (2), 98‒131, doi: 10.1002/ppap.201400052. reus and Escherichia coli. Applied and Environ- 2. JELIL, R. Abd. A review of low-temperature mental Microbiology , 2008, 74 (7), 2171‒2178, plasma treatment of textile materials. Journal of doi: 10.1128/AEM.02001-07. Material Science , 2015, 15 (18), 5913‒5943, doi: 12. GAO, Yuan, CRANSTON, Robin. Recent ad- 10.1007/s10853-015-9152-4. vances in antimicrobial treatments of textiles. 3. YIP, Joanne, LUK, Mya. Microencapsulation Textile Research Journal , 2008, 78 (1), 60‒72, doi: technologies for antimicrobial textiles. In Anti- 10.1177/0040517507082332. microbial textiles . Edited by S. Gang. Cambridge : 13. ERCEGOVIĆ RAŽIĆ, Sanja, PERAN, Jelena, KO- Woodhead Publishing, 2016, pp. 19‒46, doi: SALEC, Ivan. Functionalization of cellulose-based 10.1016/B978-0-08-100576-7.00003-1. material by surface modi cations using plasma 4. RAMACHANDRAN, T., RAJENDRAKUMAR, and organosilicone/Ag compounds. Proceedings of Ku mar, RAJENDRAN, R. Antimicrobial tex- 2nd International Conference on Natural Fibers – tiles – an overview. Journal of the Institution of from Nature to Market . Edited by R. Franqueiro. Engineers , 2004, 84 (2), 42‒47. Guimarães : University of Minho, 2015, 1‒9.

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